目的探讨METTL14通过调控巨噬细胞分化抑制宫颈癌病理性发展及相关机制。方法检测宫颈癌病变样本METTL14 m RNA和蛋白,以及IL-6、iNOS、Arg-1和CD206表达变化。PMA诱导THP-1细胞转化为巨噬细胞,慢病毒过表达或抑制METTL14表达,检测IL-6...目的探讨METTL14通过调控巨噬细胞分化抑制宫颈癌病理性发展及相关机制。方法检测宫颈癌病变样本METTL14 m RNA和蛋白,以及IL-6、iNOS、Arg-1和CD206表达变化。PMA诱导THP-1细胞转化为巨噬细胞,慢病毒过表达或抑制METTL14表达,检测IL-6、iNO、Arg-1和CD206表达变化以及PI3K/AKT/GSK3β/β-catenin信号通路相关蛋白表达情况。随后加入PI3K/AKT/GSK3β/β-catenin信号通路激动剂和抑制剂,检测过表达或抑制METTL14后,巨噬细胞IL-6、iNO、Arg-1和CD206表达变化,并取其上清制成条件培养基,孵育Hela细胞,检测细胞凋亡和增殖情况。结果1)宫颈癌病变组织中METTL14 mRNA和蛋白表达降低(P<0.05),巨噬细胞M1型标志物IL-6和iNOS表达明显降低(P<0.05),而M2型标志物Arg-1和CD206表达明显升高(P<0.05)。2)巨噬细胞过表达METTL14后,IL-6和iNOS表达明显升高(P<0.05),而Arg-1和CD206表达明显降低(P<0.05),M1/M2比例升高;抑制METTL14表达后,M1型标志物降低(P<0.05),M2型标志物升高(P<0.05),M1/M2比例降低。3)巨噬细胞中转染OE-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被抑制(P<0.05);加入PI3K/AKT激动剂后,M1型标志物降低而M2型标记物升高(P<0.05),M1/M2比例降低;OE-METTL14可逆转此趋势。Sh-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被激活(P<0.05),加入PI3K/AKT抑制剂后,M1型标志物升高而M2型标记物降低(P<0.05),M1/M2比例升高;Sh-METTL14可逆转此趋势。4)取转染OE-METTL14慢病毒后的巨噬细胞上清培养Hela细胞,可见细胞凋亡明显增多(P<0.05),增殖明显减少(P<0.05)。Sh-METTL14组的Hela细胞则表现出细胞凋亡减少(P<0.05),增殖增多(P<0.05)。结论METTL14通过PI3K/AKT/GSK3β/β-catenin信号通路调控巨噬细胞分化可能有促进宫颈癌细胞凋亡,抑制增殖的作用。展开更多
Objective: To explore whether METTL14 is involved in regulating the fibroblast proliferation and inflammatory cytokine secretion by regulating the m6A modification of pri‑miR‑141. Methods: MRC‑5 cells were transfected...Objective: To explore whether METTL14 is involved in regulating the fibroblast proliferation and inflammatory cytokine secretion by regulating the m6A modification of pri‑miR‑141. Methods: MRC‑5 cells were transfected via METTL14 overexpression lentivirus to increase METTL14 expression. Levels of METTL14 and ZEB1 were measured by qPCR and western blot. The effect of METTL14 on MRC‑5 proliferation and apoptosis was determined by CCK‑8 and flow cytometry, respectively. The ELISA kits of IL‑2, IL6 and TNF‑α were used to detect the effect of METTL14 on MRC‑5 inflammatory secretion. m6A modification site on pri‑miR‑141 was detected by meRIP. The binding site between pri‑miR‑141 and METTL14 was determined by RIP. Results: We successfully upregulated METTL14 expression in MRC‑5 cells. Elevated METTL14 promoted MRC‑5 cell proliferation, suppressed its apoptosis and promoted inflammatory factors secretion in MRC‑5 cells. pri‑miR‑141 had m6A modification sites. pri‑miR‑141 can directly bind to METTL14. METTL14 upregulation increased miR‑141 while suppressed ZEB1 expression. Conclusion: METTL14 can promote the expression of miR‑141 by increasing the m6A modification site of pri‑miR‑141, and inhibit ZEB1, thereby promoting the proliferation of fibroblasts and the secretion of inflammatory factors.展开更多
文摘目的探讨METTL14通过调控巨噬细胞分化抑制宫颈癌病理性发展及相关机制。方法检测宫颈癌病变样本METTL14 m RNA和蛋白,以及IL-6、iNOS、Arg-1和CD206表达变化。PMA诱导THP-1细胞转化为巨噬细胞,慢病毒过表达或抑制METTL14表达,检测IL-6、iNO、Arg-1和CD206表达变化以及PI3K/AKT/GSK3β/β-catenin信号通路相关蛋白表达情况。随后加入PI3K/AKT/GSK3β/β-catenin信号通路激动剂和抑制剂,检测过表达或抑制METTL14后,巨噬细胞IL-6、iNO、Arg-1和CD206表达变化,并取其上清制成条件培养基,孵育Hela细胞,检测细胞凋亡和增殖情况。结果1)宫颈癌病变组织中METTL14 mRNA和蛋白表达降低(P<0.05),巨噬细胞M1型标志物IL-6和iNOS表达明显降低(P<0.05),而M2型标志物Arg-1和CD206表达明显升高(P<0.05)。2)巨噬细胞过表达METTL14后,IL-6和iNOS表达明显升高(P<0.05),而Arg-1和CD206表达明显降低(P<0.05),M1/M2比例升高;抑制METTL14表达后,M1型标志物降低(P<0.05),M2型标志物升高(P<0.05),M1/M2比例降低。3)巨噬细胞中转染OE-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被抑制(P<0.05);加入PI3K/AKT激动剂后,M1型标志物降低而M2型标记物升高(P<0.05),M1/M2比例降低;OE-METTL14可逆转此趋势。Sh-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被激活(P<0.05),加入PI3K/AKT抑制剂后,M1型标志物升高而M2型标记物降低(P<0.05),M1/M2比例升高;Sh-METTL14可逆转此趋势。4)取转染OE-METTL14慢病毒后的巨噬细胞上清培养Hela细胞,可见细胞凋亡明显增多(P<0.05),增殖明显减少(P<0.05)。Sh-METTL14组的Hela细胞则表现出细胞凋亡减少(P<0.05),增殖增多(P<0.05)。结论METTL14通过PI3K/AKT/GSK3β/β-catenin信号通路调控巨噬细胞分化可能有促进宫颈癌细胞凋亡,抑制增殖的作用。
基金Health Industry Scientific Research Project of Hainan Province (No.21A200259)。
文摘Objective: To explore whether METTL14 is involved in regulating the fibroblast proliferation and inflammatory cytokine secretion by regulating the m6A modification of pri‑miR‑141. Methods: MRC‑5 cells were transfected via METTL14 overexpression lentivirus to increase METTL14 expression. Levels of METTL14 and ZEB1 were measured by qPCR and western blot. The effect of METTL14 on MRC‑5 proliferation and apoptosis was determined by CCK‑8 and flow cytometry, respectively. The ELISA kits of IL‑2, IL6 and TNF‑α were used to detect the effect of METTL14 on MRC‑5 inflammatory secretion. m6A modification site on pri‑miR‑141 was detected by meRIP. The binding site between pri‑miR‑141 and METTL14 was determined by RIP. Results: We successfully upregulated METTL14 expression in MRC‑5 cells. Elevated METTL14 promoted MRC‑5 cell proliferation, suppressed its apoptosis and promoted inflammatory factors secretion in MRC‑5 cells. pri‑miR‑141 had m6A modification sites. pri‑miR‑141 can directly bind to METTL14. METTL14 upregulation increased miR‑141 while suppressed ZEB1 expression. Conclusion: METTL14 can promote the expression of miR‑141 by increasing the m6A modification site of pri‑miR‑141, and inhibit ZEB1, thereby promoting the proliferation of fibroblasts and the secretion of inflammatory factors.
